Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the CCR. In FY2017, the unit collaborated in 41 different projects, with more than 1750 samples processed and analyzed. These projects are being performed in collaboration with 32 different investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, and methylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. The resource is also being used to identify protein interactors of both proteins and nucleic acids, including identification of those that change following post-translational modification. These studies help provide critical insight into protein function and regulation. Mass spectrometry is additionally being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify whole proteomes, such as the protein composition of a subcellular organelle or vesicle (e.g., mitochondria and exosomes) or a whole cell. Additionally, we are collaborating on a global quantitative phosphoproteome study, in which the global level of phosphorylation is compared. These discovery-oriented studies, which are sample- and time-intensive, provide broad information for defining new hypotheses and provide new insight into global protein activities and cellular responses. In the past year, five collaborative studies have been published, and several other projects have been completed and the corresponding manuscripts are under review. The first study, a collaboration with Dr. Paul Randazzo, Laboratory of Cellular and Molecular Biology, identified a novel interaction between Kinesin-like protein 2A (Kif2A) and Arf GAP with GTP-binding protein-like, ankyrin repeats and PH domains 1 (AGAP1) and elucidated the molecular function of this interaction. Mass spectrometry was used to screen for interactors of the GLD and PH domains of AGAP1, an Arf GAP that binds directly to the clathrin adaptor protein AP-3 to form a vesicular coat or to muscarinic receptor to modulate its trafficking. Among the identified interactors were a Ras family GTP-binding protein, actin and actin-associated proteins, and a kinesin. Yeast-2-hybrid analyses of binding partners of AGAP proteins also identified kinesin proteins as interactors, and several kinesins were shown to bind to AGAP1 by co-immunoprecipitation. Kif2A increased GAP activity of AGAP1, whereas the GLDPH domains of AGAP1 increased ATPase activity of Kif2A. Knockdown of either protein slowed cell migration and accelerated cell spreading, indicating that the Kif2A-AGAP1 complex contributes to control of cytoskeleton remodeling involved in cell movement. In this study, the initial mass spectrometry analysis to identify binding parts of AGAP1 was essential to the investigation of this family as modulators of AGAP1 activity. The study was published in the Journal of Biological Chemistry and selected as paper of the week. In the second study, mass spectrometry was used to demonstrate the utility of a new method for sorting viral particles. The laboratory of Dr. Marjorie Robert-Guroff, Vaccine Branch, used nanoscale fluorescence-activated cytometric cell sorting to sort infectious HIV from monocytes/macrophages, T cells, as well as from archived plasma. Sorted populations from human plasma were subsequently analyzed both genetically and using mass spectrometry. Although the yield of HIV was low, the mass spectrometry analysis confirmed the presence of HIV proteins and the results were consistent with the genetic analysis. Further, the analysis demonstrates the potential to sort patient populations of HIV and perform proteomics analyses with them. This study was published in JCI Insight. The third study used crosslinking-mass spectrometry analyses to demonstrate that the N- and C-terminal regions of the Vibrio cholerae DNA replication initiator protein RctB could interact in trans. RctB specifically initiates replication of V. cholerae Chromosome 2 (Chr2); RctB binds to both 12-mer sites to promote replication as well as to 39-mer sites to inhibit Chr2 replication. Additionally, binding of the chaperone DnaJK promotes both effects. Using mass spectrometry in combination with crosslinking analysis, we demonstrated that the N-terminal K-I site of RctB, which binds to DnaK, directly contacts the C-terminal winged-helix domain, which binds DNA. Other identified crosslinks were formed in regions shown to be important for dimerization and DNA binding. These results suggest that the RctB K-I site could be directly contacting the region responsible for 39-mer binding to cause autoinhibition. These findings, performed in collaboration with Dr. Dhruba Chattoraj, Laboratory of Biochemistry and Molecular Biology, were published in mBio. We have further used mass spectrometry to characterize the protein interactors of PINCR, a p53-regulated long noncoding RNA (lncRNA). PINCR is an 2.2 kb spliced intergenic lncRNA transcribed from the X-chromosome that was observed to be induced approximately 100-fold following DNA damage in a p53-dependent manner. Further, PINCR was found to exert a prosurvival function in human colorectal cancer cells (CRC) in vitro and to promote tumor growth in vivo. Mass spectrometry analysis was used to identify the protein interactors of PINCR following pulldown of a biotinylated form of the RNA. Among those interactors, we identified the RNA-binding protein Matrin 3, which in turn associates with p53. Through its interaction with Matrin 3, and thereby working also through p53, PINCR induces the expression of a subset of p53 target genes, including BTG2, GPX1 and RRM2B. This study demonstrates the presence of a critical prosurvival function of a p53/PINCR/Matrin 3 comeplex in response to DNA damage in CRC cells. This research, performed in collaboration with Dr. Ashish Lal, Genetics Branch, was published in eLIFE. Finally, we worked with Dr. Pascale Legault, Universite de Montreal, to develop a novel methodology to pulldown and identify the protein interactors of RNAs. In the method, specific RNA molecules are in vitro transcribed with an ARiBo tag, which contains an activatable glmS ribozyme and the BoxB RNA from bacteriophage lambda. The lambda boxB RNA allows immobilization of the RNA on glutathione sepharose via its high affinity to a recombinant lambda N-GST fusion protein, whereas the glmS ribozyme can be activated by glucosamine-6-phosphate to free the RNA of interest from the resin. Thus, the RNA of interest can be immobilized on resin for incubation with cell extract and pulldown, then the RNA-protein complexes eluted by activation of the ribozyme. Protein interactors are then identified by mass spectrometry analysis. To demonstrate the utility of the method, we identified the protein interactome of two members of the let-7 family of miRNAs, let-7a1 and let-7g. Mass spectrometry analysis demonstrated the specific enrichment of several known interactors of both miRNAs, including lin28A and lin28B with low background binding to the ARiBo tag alone. Thus, this method represents a new means to identify the protein interactors of specific RNA molecules. This research was published in RNA.